Raman scattering can result from an inelastic collision of a photon with atoms or molecules. During elastic collisions (Rayleigh scattering), an atom can be excited from a ground state to a higher energy state, and can then relax back to the original ground state upon which the atom emits a photon at the same frequency as light incident on the atom. However, during an inelastic collision, an excited atom may relax to a vibrationally excited state rather than the ground state upon which a scattered photon can be emitted (Stoke's line) with energy lower than the incident photon. If the incident photon interacts with an already vibrationally excited molecule, the scattered photon can be emitted with higher energy (Anti-Stokes line) than the incident photon. Illustrated in FIG. 4 is an embodiment of a representation of the Raman spectrum.
Raman spectroscopy can give information about the characteristic vibrational states of the chemical bonds of the molecules being studied. This molecular level specificity has made Raman spectroscopy a widely used spectroscopic tool for the determination of molecular structure and for compound identification. As such, this technique is useful for a variety of applications that require the detection of biologically significant molecules such as toxins and disease biomarkers.
Despite the inherent advantages of using Raman Spectroscopy, its usage has been somewhat limited because it is an inefficient analysis technique. Raman spectroscopy has a small scatter cross section compared to Fluorescence spectroscopy (10−30 cm2 per molecule when compared to 10−16 cm2 for fluorescence.) Thus, Raman spectroscopy often cannot be used to analyze compounds of biological significance due to the generally low concentration of analytes in biological samples. There are, however, ways to greatly enhance the Raman signal by using particularly structured metallic (e.g. Ag, Au, and Cu) substrates, where the metallic structure enhances the Raman scattering. Surface Enhanced Raman Scattering (SERS) was first reported in 1974 by Fleischmann. M. Fleischmann, P. J. Hendra, and A. J. McQuillan, “Raman spectra of pyridine adsorbed at a silver electrode,” Chemical Physics Letters 26, 163-166 (1974). Fleischmann observed a large enhancement of Raman signals of pyridine molecules adsorbed on electrochemically roughened silver electrodes. SERS amplification factors (AF) of 106˜1016 have been achieved using a wide range of SERS substrates, thus the SERS enhancement effect has made Raman spectroscopy an increasingly important analytical tool in the biological sciences.
Typical SERS substrates are fabricated using methods that result in noble metal nanostructures stochastically distributed over a substrate surface, e.g. electrochemically roughened electrodes, sputtered films, chemically etched films, electroless deposited films, and colloidal metal particles. Another way to obtain large amplification in Raman scattering can be to place a substrate in close proximity of a sharp metallic tip. Although SERS amplifications of 103-105 have been reported using such substrates, these substrates are often not reproducible.
Still other SERS substrates are created by depositing colloidal silver particles on quartz/glass substrates using standard wet chemistry. These fabrication methods can typically result in a monolayer of nanoparticles. Using a process such as depositing the particles on quartz or glass using standard wet chemistry can create hot spots during the colloidal preparation.
SERS substrates can also be fabricated by controlling the pattern of the nanostructures on a substrate using electron-beam lithography. Substrates created using this method are now commercially available and manufactured by D3 Technologies Ltd, Glasgow, UK. These substrates are fabricated using a multi-step process that results in substrates which are quite expensive (e.g. $75-$125/substrate.) These substrates are also usually small in size (e.g. 4 mm by 4 mm.) There is no easy way to commercialize the method described above because SERS substrates created using the nano-lithographic process can have the following limitations: they are expensive to produce; the equipment required to produce them is sophisticated and expensive; and it is difficult to produce substrates that have a size which exceeds approximately a centimeter square.
There exists a need for inexpensive methods and compositions for fabricating analytical substrates for use in SERS and substrates that reliably amplify incident photons emitted by a spectrometer. In particular there exists a need for cost effective SERS substrate fabrication methods that produce analytical substrates that meet a particular performance measurement.